Guidelines for the diagnosis and management of hereditary spherocytosis

Hereditary spherocytosis (HS) is a heterogeneous group of disorders with regard to clinical severity, protein defects and mode of inheritance. It is relatively common in Caucasian populations; most affected individuals have mild or only moderate haemolysis. There is usually a family history, and a typical clinical and laboratory picture so that the diagnosis is often easily made without additional laboratory tests. Atypical cases may require measurement of erythrocyte membrane proteins to clarify the nature of the membrane disorder and in the absence of a family history, occasionally molecular genetic analysis will help to determine whether inheritance is recessive or non-dominant. It is particularly important to rule out stomatocytosis where splenectomy is contraindicated because of the thrombotic risk. Mild HS can be managed without folate supplements and does not require splenectomy. Moderately and severely affected individuals are likely to benefit from splenectomy, which should be performed after the age of 6 years and with appropriate counselling about the infection risk. In all cases careful dialogue between doctor, patient and the family is essential. Laparoscopic surgery, when performed by experienced surgeons, can result in a shorter hospital stay and less pain.     

Membrane protein pattern in hereditary spherocytosis in five subjects from north-east Italy obtained by SDS-PAGE using N,N'-diallyltartardiamide

In the present study we examined five subjects affected by hereditary spherocytosis (three unsplenectomized and two splenectomized), coming from an area in the north-east of Italy where hereditary spherocytosis is an anaemic disease with very low incidence. All patients showed a low degree of spectrin deficiency (14%), detected with sodium dodecyl sulfate polyacrylamide gel electrophoresis. Moreover, when this analysis was performed with N,N'-diallyltartardiamide as cross-linking agent instead of N,N'-methylenbisacrylamide, some unusual bands appeared in the region between proteins 4.2 and 5, the three unsplenectomized and two splenectomized patients showing different patterns. We hypothesise that some alterations of proteins in this region (e.g. the 4.5 or 4.9 bands), possibly due to proteolysis, must have occurred in relation to the disease.     

Amino-acid substitution in α-spectrin commonly coinherited with nondominant hereditary spherocytosis

Nondominant hereditary spherocytosis (ndHS) is a disorder characterized in some patients by severe hemolytic anemia and marked deficiency of erythrocyte spectrin. This report describes the identification of a variant spectrin chain, α-spectrin Bughill or α^BH, that is associated with this disorder in a number of patients. Tryptic maps of spectrin from affected individuals revealed an acidic shift in isoelectric point of the αll domain peptides at 46 kD and 35 kD. A point mutation at codon 970 of the α-spectrin gene (GCT→GAT), that changes the encoded amino acid from an alanine to an aspartic acid, was identified in genomic DNA of affected patients. The α^BH variant was present in 8 patients with ndHS from five different kindreds but was absent in 4 patients from two other kindreds. The 8 ndHS patients with the α^BH variant appeared to be homozygous for the α^BH variant by analysis of peptide maps of limited tryptic digests of erythrocyte spectrin. However, following genomic DNA analysis, only 2 of these patients were true homozygotes, whereas 6 were found to be doubly heterozygous for the α^BH allele and a second, presumably abnormal, α-spectrin gene. These results suggest that, in these 6 patients, the second α-spectrin allele is in fact associated with one or more genetic defect(s), causing decreased accumulation of α-spectrin. The pattern of transmission of the α^BH allele in certain families suggests that the α^BH amino-acid substitution is not itself responsible for ndHS but is more likely a polymorphic variant that, in some but not all cases, is in linkage disequilibrium with another uncharacterized α-spectrin gene defect that itself is a cause of ndHS. Am. J. Hematol. 54:233–241, 1997 © 1997 Wiley-Liss, Inc.     

A variant of the EPB3 gene of the anti-Lepore type in hereditary spherocytosis

The EPB3 gene encodes band 3 (anion exchanger 1) of the red cell membrane. A subset of hereditary spherocytosis (HS) is associated with EPB3 gene mutations and band 3 deficiency. We report a large Italian family in which 10 of the 27 members investigated displayed an autosomal dominant HS. SDS-PAGE revealed a reduction in band 3 in the patients. Screening of the Pst I polymorphic site confirmed the linkage of HS with the EPB3 gene. Analysis of complementary and genomic DNA showed a large additional segment. Nucleotide sequencing disclosed an in-frame duplication of 69 nucleotides (nt) including a triplet of intronic origin and a genuine exonic duplication of 66 nt. Two CCTGC sequences occurred close to one another, one near the intron 12 acceptor splice site (nt −7 to −3), and the other within exon 13 (nt 1494–1498). We assumed that the abnormal allele arose from an unequal recombination event of the anti-Lepore type between the two CCTGC sequences.
At the level of the mutated protein, termed band 3 Milano, the additional segment (Gln plus duplication of residues 478–499) corresponded to the last part of the third transmembrane domain (TM3), the entire second outer loop and part of TM4 as it is currently defined in hydropathy analysis. After deglycosylation of band 3, only the normal band was detected, supporting the view that band 3 Milano is probably not incorporated into the membrane.     

Abnormal binding of spectrin to the membrane of erythrocytes in some cases of hereditary spherocytosis

Summary In two cases of hereditary spherocytosis that we have examined, spectrin was bound abnormally tightly to the erythrocyte membrane, and could not be released by low ionic strength dialysis. This type of behaviour occurs in normal red cells only after heating above 50 C. It appears that some cases of spherocytosis may be due to the presence of a protein which is abnormally temperature sensitive.This work was supported by an Australian Government Research Grant     

Role of the interaction between Lu/BCAM and the spectrin‐based membrane skeleton in the increased adhesion of hereditary spherocytosis red cells to laminin

Lu/BCAM, the unique erythroid receptor for laminin 511/521, interacts with the erythrocyte membrane skeleton through spectrin binding. It has been reported that Hereditary Spherocytosis red blood cells (HS RBC) exhibit increased adhesion to laminin. We investigated the role of Lu/BCAM–spectrin interaction in the RBC adhesion properties of 2 splenectomised HS patients characterized by 40% spectrin deficiency. Under physiological flow conditions, HS RBC exhibited an exaggerated adhesion to laminin that was completely abolished by soluble Lu/BCAM. Triton extraction experiments revealed that a greater fraction of Lu/BCAM was unlinked to the membrane skeleton of HS RBC, as compared to normal RBC. Disruption of the spectrin interaction site in Lu/BCAM expressed in the transfected K562 cell line resulted in a weakened interaction to the skeleton and an enhanced interaction to laminin. These results demonstrated that the adhesion of HS RBC to laminin was mediated by Lu/BCAM and that its interaction with the spectrin‐based skeleton negatively regulated cell adhesion to laminin. Finally, the results of this study strongly suggest that the reinforced adhesiveness of spectrin‐deficient HS RBC to laminin is partly brought about by an impaired interaction between Lu/BCAM and the membrane skeleton.     

Hereditary spherocytosis is associated with decreased pyruvate kinase activity due to impaired structural integrity of the red blood cell membrane

Hereditary spherocytosis (HS) is characterised by increased osmotic fragility and enhanced membrane loss of red blood cells (RBC) due to defective membrane protein complexes. In our diagnostic laboratory, we observed that pyruvate kinase (PK) activity in HS was merely slightly elevated with respect to the amount of reticulocytosis. In order to evaluate whether impaired PK activity is a feature of HS, we retrospectively analysed laboratory data sets from 172 unrelated patients with HS, hereditary elliptocytosis (HE), glucose-6-phosphate dehydrogenase (G6PD) or PK deficiency, sickle cell or haemoglobin C disease, or β-thalassaemia minor. Results from linear regression analysis provided proof that PK activity decreases with rising reticulocyte counts in HS (R² = 0·15; slope = 9·09) and, less significantly, in HE (R² = 0·021; slope = 8·92) when compared with other haemolytic disorders (R² ≥ 0·65; slopes ≥ 78·6). Reticulocyte-adjusted erythrocyte PK activity levels were significantly lower in HS and even declined with increasing reticulocytes (R² = 0·48; slope = −9·74). In this report, we describe a novel association between HS and decreased PK activity that is apparently caused by loss of membrane-bound PK due to impaired structural integrity of the RBC membrane and may aggravate severity of haemolysis in HS.     

Genetic mapping of the multiple endocrine neoplasia type 1 locus at 11q13

Abstract. Oncogenesis of tumours related to multiple endocrine neoplasia type 1 (MEN1) is associated with somatic deletions involving the MEN1 locus at chromosomal region 11q13, suggesting inactivation of a tumour-suppressor gene in this region. Here we describe the localization of the MEN1 gene to a 900-kb region, based on linkage analysis in affected families and deletion mapping of MEN1-associated tumours. In addition, a set of microsatellite markers mapped to the 11q11–13 region were used for linkage analysis in a large Tasmanian MEN1 pedigree, demonstrating the usefulness of these markers for presymptomatic testing in affected families.     

Severe HDL deficiency due to novel defects in the ABCA1 transporter

Objectives. The objective was the identification and functional characterization of mutations in the ABCA1 gene in four patients with severe HDL deficiency. Subjects. Patients were referred to the clinic because of almost complete HDL deficiency. Methods. The ABCA1 gene was sequenced directly. The analysis of the ABCA1 protein, ABCA1 mRNA and ABCA1‐mediated cholesterol efflux was performed in cultured fibroblasts. Intracellular localization of ABCA1 mutants was investigated in transfected HEK293 cells. Results. Two patients were homozygous for mutations in the coding region of the ABCA1 gene, resulting in an amino acid substitution (p.A1046D) and a truncated protein (p.I74YFsX76). The third patient was homozygous for a splice site mutation in intron 35 (c.4773 + 1g>a), resulting in an in‐frame deletion of 25 amino acids (del p.D1567_K1591) in ABCA1. These patients had clinical manifestations of accumulation of cholesterol in the reticulo‐endothelial system. The fourth patient, with preclinical atherosclerosis, was a compound heterozygote for two missense mutations (p.R587W/p.W1699C). ABCA1‐mediated cholesterol efflux was abolished in fibroblasts from patients with p.A1046D and del p.D1567_K1591 mutants and in fibroblasts homozygous for p.R587W. A reduced ABCA1 protein content was observed in these cells, suggesting an increased intracellular degradation. The mutant p.W1699C was largely retained in the endoplasmic reticulum, when expressed in HEK293 cells. Conclusions. The homozygotes for mutations which abolish ABCA1 function showed overt signs of involvement of the reticulo‐endothelial system. This was not the case in the compound heterozygote for missense mutations, suggesting that this patient retains some residual ABCA1 function that reduces cholesterol accumulation in the reticulo‐endothelial system.